Pathogenic LRRK2 regulates centrosome cohesion via Rab10/RILPL1-mediated CDK5RAP2 displacement

Summary Mutations in LRRK2 increase its kinase activity and cause Parkinson's disease. LRRK2 phosphorylates a subset of Rab proteins which allows for their binding to RILPL1. The phospho-Rab/RILPL1 interaction causes deficits in ciliogenesis and interferes with the cohesion of duplicated centrosomes. We show here that centrosomal deficits mediated by pathogenic LRRK2 can also be observed in patient-derived iPS cells, and we have used transiently transfected cell lines to identify the underlying mechanism. The LRRK2-mediated centrosomal cohesion deficits are dependent on both the GTP conformation and phosphorylation status of the Rab proteins. Pathogenic LRRK2 does not displace proteinaceous linker proteins which hold duplicated centrosomes together, but causes the centrosomal displacement of CDK5RAP2, a protein critical for centrosome cohesion. The LRRK2-mediated centrosomal displacement of CDK5RAP2 requires RILPL1 and phospho-Rab proteins, which stably associate with centrosomes. These data provide fundamental information as to how pathogenic LRRK2 alters the normal physiology of a cell.


INTRODUCTION
Point mutations in Leucine rich repeat kinase 2 (LRRK2) cause autosomal-dominant familial Parkinson's disease (PD). Pathogenic LRRK2 phosphorylates a subset of Rab proteins (Steger et al., 2016), small GTPases which function as key regulators for intracellular membrane trafficking events (Hutagalung and Novick, 2011;Pfeffer, 2013). Rab8 and Rab10 act as prominent LRRK2 kinase substrates, and upon their phosphorylation both gain the ability to bind to RILPL1 . Pathogenic LRRK2 blocks primary cilia formation in cultured cells and in certain areas of the mouse brain, which is associated with the RILPL1mediated centrosomal accumulation of phospho-Rab10 (Dhekne et al., 2018;Steger et al., 2017). This accumulation interferes with the recruitment of tau tubulin kinase 2 (TTBK2), thereby preventing the release of centriolar CP110 which is necessary for the initiation of cilia formation (Sobu et al., 2021).
We have previously shown that the LRRK2-mediated phosphorylation of Rab8 and Rab10 and their subsequent RILPL1-mediated centrosomal accumulation not only interferes with ciliogenesis, appropriate centrosome positioning and directional cell migration, but also with the cohesion of duplicated centrosomes in dividing cells (Lara Ordonez et al., 2019;Madero-Pé rez et al., 2018). In peripheral cells from LRRK2-PD patients as compared to healthy controls, quantitative mass spectrometry-based assays can detect increased phospho-Rab10 levels (Karayel et al., 2020), and centrosomal cohesion deficits serve as a sensitive cellular biomarker for enhanced LRRK2 kinase activity (Fernandez et al., 2019). However, neither of those assays can be easily translated into clinical settings, requiring the need for further mechanistic insights into these LRRK2 kinase-mediated cellular alterations.
Duplicated centrosomes gradually mature during the G2 phase of the cell cycle and remain connected by a proteinaceous linker until the onset of mitosis to assure appropriate centrosome cohesion (Agircan et al., 2014;Mardin and Schiebel, 2012). The filamentous linker proteins rootletin and cep68 hold the duplicated centrosomes together by binding to the centrosomal docking protein c-Nap1 (Bahe et al., 2005;Graser et al., 2007;Vlijm et al., 2018;Yang et al., 2006). At the onset of mitosis, the mitotic kinase Polo-like kinase 1 (Plk1) activates Nek2a, which in turn phosphorylates linker and docking components to cause linker The LRRK2-mediated centrosomal cohesion deficits depend on both the GTP conformation and phosphorylation status of Rab8a The LRRK2-mediated phosphorylation of Rab8a (on T72) and Rab10 (on T73) allows for their interaction with a new set of effector proteins including RILPL1 , which causes their accumulation at/around the centrosome and correlates with the centrosomal cohesion and ciliogenesis deficits (Dhekne et al., 2018;Lara Ordonez et al., 2019;Madero-Pé rez et al., 2018). Recent studies indicate that such interactions may also depend on the GTP-bound conformation of the phosphorylated Rab proteins (Waschbü sch et al., 2020). We therefore analyzed whether the LRRK2-mediated centrosomal cohesion deficits were dependent on both the GTP conformation and phosphorylation status of Rab8a. We coexpressed FLAG-tagged wild-type LRRK2 with either GFP-tagged wild-type Rab8a, Rab8a-Q67L (GTP-trapped conformation) or Rab8a-T22N (GDP-bound conformation) in HEK293T cells, respectively. As previously described , co-expression of FLAG-tagged wild-type LRRK2 with GFP-tagged wild-type Rab8a to increase the amount of phosphorylated-tagged Rab8a caused a centrosomal cohesion phenotype (Figures 2A and 2B). Co-expression of FLAG-tagged wild-type LRRK2 with GFP-tagged Rab8a-Q67L also caused a pronounced cohesion deficit, whereas no effects were observed when coexpressing Rab8a-T22N or a GTP-trapped Rab8a mutant unable to be phosphorylated by LRRK2 (Rab8a-Q67L-T72A) (Figures 2A and 2B). The centrosomal deficits mediated by co-expression of FLAG-tagged LRRK2 with GFP-tagged wild-type or GTP-trapped Rab8a correlated with an increase in the phosphorylation status of the transiently expressed tagged Rab8a proteins ( Figure 2C) and were at least in part reverted by the LRRK2 kinase inhibitor MLi2 ( Figure 2B). In addition, all GFP-tagged mutants except the GDP-bound version of Rab8a were expressed to similar degrees and were properly membrane-associated ( Figures 2C and 2D). These data indicate that the LRRK2-mediated centrosomal cohesion deficits depend on both the phosphorylation and nucleotide-bound status of Rab8a.
We wondered whether co-expression of GTPase activating proteins (GAPs) to decrease the GTP-bound status of the endogenous phospho-Rab proteins may reduce the cohesion deficits mediated by pathogenic LRRK2. TBC1D4/AS160 displays GAP activity toward Rab10 in vivo (Sano et al., 2007), and both ll OPEN ACCESS iScience 25, 104476, June 17, 2022 iScience Article TBC1D17 and TBC1D30 have been reported to display GAP activity toward Rab8a Yoshimura et al., 2007). Co-expression of GFP-tagged TBC1D30 did not alter the centrosomal cohesion deficits mediated by FLAG-tagged pathogenic LRRK2 ( Figures S3A and S3B). Similarly, expression of active or inactive GFP-tagged TBC1D4 or TBC1D17 constructs only marginally decreased the pathogenic LRRK2mediated centrosomal cohesion deficits ( Figures S3A and S3C). None of the three TBC proteins were able to decrease the LRRK2-mediated phosphorylation levels of endogenous Rab8a or Rab10 as assessed by Western blotting (Figure S3D) or the localization of phospho-Rab10 as assessed by immunocytochemistry Figure 1. Pathogenic LRRK2 causes ciliary and centrosomal cohesion deficits in patient-derived iPSCs (A) Example of undifferentiated healthy patient-derived LRRK2 WT/WT (control) or gene-edited LRRK2 R1441C/WT (R1441C) iPSCs stained for Arl13b (red) and DAPI (blue). Arrows point to elongated, Arl13b-positive cilia. Scale bar, 10 mm. (B) Quantification of the percentage reduction in ciliated cells in control and gene-edited R1441C iPSCs. Around 200 cells were quantified per experiment. Bars represent mean G SEM (n = 3 independent experiments; p = 0.01); *p < 0.05. (C) Example of control or R1441C iPSCs treated in the absence or presence of MLi2 (200 nM, 2 h), and stained for g-tubulin (green), pericentrin (red) and DAPI (blue). Arrows point to centrosomes as identified by co-staining with the two centrosomal markers. Scale bar, 10 mm. (D) Quantification of the percentage of control or R1441C iPSCs with split centrosomes (duplicated centrosomes with a distance between their centers >1.5 mm), in the presence or absence of 200 nM MLi2 for 2 h before immunocytochemistry as indicated. Around 150-200 cells were quantified per condition per experiment. Bars represent mean G SEM (n = five to seven independent experiments; ctrl versus R1441C-LRRK2, p = 0.001; R1441C-LRRK2 versus R1441C-LRRK2 + MLi2: p = 0.002); ***p < 0.005. (E) Same as in (D), but depicting the mean distance between duplicated centrosomes. Around 50 cells with duplicated centrosomes per condition were analyzed. Bars represent mean G SEM (ctrl versus R1441C-LRRK2, p = 0.002; R1441C-LRRK2 versus R1441C-LRRK2 + MLi2: p = 0.024); ***p < 0.005; *p < 0.05.
Phospho-Rab8a and phospho-Rab10 accumulate on centrosomes in the presence of pathogenic LRRK2 To determine whether the phospho-Rabs stably interact with centrosomes, we biochemically purified fractions enriched for centrosomes from HEK293T cells. Centrosomes were isolated by centrifugation using a discontinuous sucrose gradient, and fractions were immunoblotted for centrosomal markers, with the centrosome fraction eluting at around 55% sucrose as previously described (Gogendeau et al., 2015) (Figure S4A). To enhance detection of the Rab proteins, we transiently expressed FLAG-tagged Rab8a or FLAG-tagged Rab10. Although neither total nor phosphorylated Rab8a were detectable on centrosomeenriched fractions in the absence of LRRK2 expression, co-expression of FLAG-Rab8a with pathogenic LRRK2 resulted in the presence of phospho-Rab8a on centrosomes ( Figure S4B). Similarly, phospho-Rab10 was detected on centrosomes in the presence, but not in the absence of pathogenic LRRK2 expression ( Figure S4C). Although the identity of the Rab8a/10-containing vesicular compartment in contact with the centrosome remains unknown, these data are consistent with the notion that both membrane-bound Rab8a and Rab10 stably associate with centrosomes upon their LRRK2-mediated phosphorylation. (A) Example of HEK293T cells co-transfected with FLAG-tagged wild-type LRRK2 and GFP-tagged Rab8a-Q67L (GTP-trapped) or GFP-tagged Rab8a-Q67L-T72A (GTP-trapped but non-phosphorylatable), and stained with antibody against FLAG (red) and the centrosomal marker pericentrin (pseudocolored blue) and DAPI. Arrows point to centrosomes in transfected cells. Cell boundaries (yellow) are shown and were determined by FLAG staining because of FLAGtagged wild-type LRRK2 expression (or GFP because of GFP-tagged Rab8a expression for single transfection experiments). Scale bar, 10 mm.
(D) HeLa cells were transfected with the indicated GFP-tagged Rab8a constructs, briefly fixed, washed and stained with DAPI. All GFP-tagged Rab8a constructs except the inactive T22N mutant display a localization consistent with their presence in a tubular early recycling compartment. Scale bar, 10 mm. iScience Article Regulation of LRRK2-mediated centrosomal cohesion deficits by the Plk1/Nek2/linker cascade We next wondered whether the centrosomal cohesion deficits are subject to regulation by linker proteins known to hold duplicated centrosomes together in a manner dependent on their phosphorylation status (Agircan et al., 2014;Mardin and Schiebel, 2012). Transient overexpression of Nek2a or treatment of cells with the phosphatase inhibitor calyculin A caused an increase in the percentage of cells with split duplicated centrosomes ( Figures S5A and S5B). This was associated with an increase in the percentage of transfected cells in G2/M phase and a concomitant decrease of cells in G1 phase ( Figure S5C), even though not leading to cell cycle arrest, as previously described (Faragher and Fry, 2003;Fry et al., 1998b;Mayor et al., 2000). Similarly, transient overexpression of distinct pathogenic LRRK2 mutants caused an increase in the percentage of transfected cells in G2/M and a decrease in G1 phase ( Figures S5D-S5F), and specifically an increase in the percentage of cells in G2 as assessed by cyclinB staining ( Figure S5G). Such cell cycle alterations were observed with pathogenic LRRK2, but not with a kinase-inactive point mutant, suggesting that they are mediated by the LRRK2 kinase activity ( Figure S5H). The cohesion deficit mediated by the expression of various pathogenic LRRK2 mutants was fully reverted when coexpressing the linker protein rootletin ( Figures 3A-3D). Furthermore, co-expression of wild type or constitutively active PP1a (Xu et al., 2003), or co-expression of inactive, dominant-negative Nek2a (Meraldi and Nigg, 2001) reverted the centrosomal cohesion deficit mediated by distinct pathogenic LRRK2 mutants ( Figures 3A-3D), whereas these constructs were without effect when expressed on their own ( Figures S6A-S6C). In A549 cells, where overexpression levels are more modest as compared to HEK293T cells ( Figure S7A), FLAG-tagged pathogenic LRRK2 expression also caused centrosomal cohesion deficits, which were partially reverted when co-expressing these constructs ( Figures S7B-S7D). In addition, acute treatment of cells with a Plk1 inhibitor partially reverted the centrosomal cohesion deficits induced by either Nek2a or by pathogenic LRRK2 expression ( Figures S8A-S8C), whereas inhibitors of p38, a member of the mitogen-activated protein kinase (MAPK) family and a regulator of the G2/M transition  were without effect ( Figures S8D and S8E). Thus, the centrosomal cohesion deficits in pathogenic LRRK2-expressing cells are not merely a reflection of cell cycle alterations but are subject to regulation by the well-known cascade involving Plk1, Nek2a, and the proteinaceous linker between duplicated centrosomes.

Pathogenic LRRK2 causes centrosomal displacement of CDK5RAP2
Because Nek2a-mediated phosphorylation of centrosomal linker proteins causes their displacement followed by centrosome separation (Bahe et al., 2005;Yang et al., 2006), we reasoned that pathogenic LRRK2 may function by a similar mechanism. Surprisingly, although Nek2a overexpression was associated with pronounced loss of rootletin staining in transfected cells (Figures S9A and S9B), this was not observed in cells expressing pathogenic LRRK2 (Figures 4A and 4B). Similarly, Nek2a expression caused pronounced loss of cep68 ( Figures S9A and S9B), another centrosomal linker protein which is part of the rootletin network (Graser et al., 2007), but only a slight displacement was observed upon pathogenic LRRK2 expression ( Figures 4A and 4B). Co-staining with a centrosomal marker confirmed that these proteins were normally localized to centrosomes in non-transfected as well as in LRRK2-transfected cells ( Figure S10A).
Apart from structural linker proteins, an RNAi screen also identified CDK5RAP2 as being required for proper centrosome cohesion (Graser et al., 2007). CDK5RAP2 is not implicated in forming the linker structure, but displays centrosomal localization throughout mitosis and is thought to influence linker integrity indirectly by a currently unknown mechanism (Agircan et al., 2014;Graser et al., 2007). Strikingly, although expression of Nek2a did not alter CDK5RAP2 staining ( Figures S9A and S9B), pathogenic LRRK2 caused the dispersal/disappearance of CDK5RAP2 staining in around 80% of transfected cells, which was reversed upon acute treatment of cells with the LRRK2 inhibitor MLi2, indicating that it was LRRK2 kinase activitymediated ( Figures 4A and 4B). Co-staining with a centrosomal marker confirmed that CDK5RAP2 was displaced from centrosomes also as judged by measuring integrated fluorescence density ( Figure S10B). In contrast, staining for the pericentriolar matrix protein pericentrin was present in virtually all interphase cells, whether transfected with Nek2 or with pathogenic LRRK2, respectively ( Figures S9, 4 and S10). The LRRK2-mediated displacement/dispersal of CDK5RAP2 was also detected when staining with another anti-CDK5RAP2 antibody ( Figure S11). In addition, displacement of CDK5RAP2 was observed in nearly 70% of A549 cells transfected with FLAG-tagged pathogenic LRRK2 and was reverted upon MLi2 treatment, indicating that it was neither cell-type nor tag-dependent (Figures S12A and S12B iScience Article immunoblot analysis (Figures S7A and S12C), CDK5RAP2 levels were not affected by pathogenic LRRK2 expression, suggesting that the relative inability to detect CDK5RAP2 by immunofluorescence is because of its dissociation from centrosomes rather than its degradation.
As another means to determine whether pathogenic LRRK2 expression interferes with the proper centrosomal localization of CDK5RAP2, we analyzed the spatial proximity between endogenous CDK5RAP2 and pericentrin by performing in situ proximity ligation assays (PLA), which allow for the sensitive detection of endogenous protein interactions (Sö derberg et al., 2006). Pathogenic LRRK2 expression led to a significant decrease in the percentage of cells displaying a proximity ligation signal between CDK5RAP2 and pericentrin ( Figures 5A and 5B), which correlated with the centrosomal displacement of CDK5RAP2 ( Figure 5C) and with the prominent centrosomal accumulation of phosphorylated Rab10, in a manner reverted upon MLi2 iScience Article treatment ( Figure 5D). We next tested whether the LRRK2-mediated CDK5RAP2 displacement requires endogenous Rab10 and RILPL1 proteins. Pathogenic LRRK2 expression caused prominent loss of endogenous CDK5RAP2 staining in A549 wild-type cells but not in cells deficient in either RILPL1 or Rab10 ( Figures 6A and 6B), indicating that it causes the centrosomal cohesion deficits by CDK5RAP2 displacement via Rab phosphorylation and RILPL1 binding. Finally, we evaluated whether expression of CDK5RAP2 may modulate the pathogenic LRRK2-mediated centrosomal cohesion deficits. Over 95% of transfected A549 wild-type cells displayed co-expression of pathogenic LRRK2 and myc-tagged CDK5RAP2, which was localized to the cytosol but also accumulating at the centrosome in the majority of co-transfected cells ( Figure 7A). Co-expression of CDK5RAP2 with pathogenic LRRK2 reverted the LRRK2-mediated centrosomal cohesion deficits both as assessed by measuring the percentage of split duplicated centrosomes or the mean distance between duplicated centrosomes ( Figures 7C  and 7D). Altogether, these results indicate that pathogenic LRRK2 triggers Rab10 phosphorylation and To probe for the observed phenotypes in cells expressing endogenous levels of LRRK2, we employed mouse embryonic fibroblasts (MEFs) derived from homozygous R1441C-LRRK2 knockin mice and littermate iScience Article matched wild-type controls . As compared to wild-type MEFs, R1441C-LRRK2 MEFs displayed an increase in G2 and a concomitant decrease in G1 phase of the cell cycle ( Figures 8A-8C). The R1441C-LRRK2 MEFs displayed a centrosomal cohesion deficit as assessed by an increase in the mean distance between duplicated centrosomes, which was reverted upon MLi2 treatment ( Figures 8D-8F). These alterations correlated with a kinase activity-mediated increase in the levels of phospho-Rab10, without changes in the total levels of CDK5RAP2 as judged by immunoblotting ( Figures 8G  and 8H).
The antibody suitable for detection of murine CDK5RAP2 in MEF cells was not compatible with immunocytochemistry approaches to probe for alterations in the centrosomal localization of CDK5RAP2. Therefore, we next employed primary human dermal fibroblasts from healthy control and G2019S LRRK2-PD patients (Madero-Pé rez et al., 2018). Centrosomal cohesion deficits were observed in primary fibroblasts from G2019S LRRK2-PD patients as compared to healthy controls, and were reverted by short-term application of MLi2 ( Figure 9A). A significant percentage of G2019S LRRK2-PD fibroblasts displayed a kinase-mediated loss of centrosomal CDK5RAP2 as compared to healthy controls ( Figures 9B and  9C), which correlated with a modest nonsignificant increase in the levels of phospho-Rab10, without changes in the total levels of CDK5RAP2 as judged by immunoblotting ( Figures 9D and 9E). Thus, endogenous levels of pathogenic LRRK2 also cause centrosomal cohesion deficits and displacement of centrosomal CDK5RAP2.

DISCUSSION
We show here that the centrosomal cohesion deficits mediated by pathogenic LRRK2 depend on the GTP conformation and phosphorylation status of membrane-bound Rab8a. A Rab8a mutant mimicking the GDP-bound state is cytosolic, unable to be phosphorylated and unable to trigger the LRRK2-mediated centrosomal cohesion deficits. Conversely, a non-phosphorylatable Rab8a mutant mimicking the GTP-bound conformation is membrane-bound yet unable to trigger the LRRK2-mediated cohesion deficits. These data indicate that the Rab protein needs to be membrane-bound and GTP-bound to be phosphorylated by LRRK2 and cause the centrosomal cohesion deficits. However, overexpression of GAP proteins for either Rab8 or Rab10 did not reverse the LRRK2-mediated Rab phosphorylation or iScience Article centrosomal cohesion deficits. These data are consistent with structural studies predicting a steric clash mediated by the phospho-residue on Rab8a which impairs its interaction with GAPs (Waschbü sch et al., 2020), and with experimental data showing that substitutions which mimic the LRRK2 phosphorylation site in Rab10 interfere with its interaction with the GAP TBC1D4/AS160 (Liu et al., 2018). Thus, strategies aimed at decreasing the GTP-bound status of membrane-bound Rab8/10 are unable to modulate the downstream effects of LRRK2-phosphorylated Rab8/10.
Rootletin forms a proteinaceous linker which holds the duplicated centrosomes together and which is displaced upon Plk1-mediated Nek2a activation and phosphorylation. We show here that overexpression of this linker protein reverts the LRRK2-mediated centrosomal cohesion deficits. In addition, overexpression of a dominant-negative version of Nek2a or overexpression of PP1a to revert the Nek2a-mediated linker phosphorylation reverts the centrosomal cohesion deficits mediated by pathogenic LRRK2. However, and in contrast to Nek2a expression, pathogenic LRRK2 does not cause the displacement of centrosomal linker proteins. These data indicate that although the Nek2a-linker cascade can modulate the centrosomal cohesion deficits mediated by pathogenic LRRK2, it is not its primary target. Indeed, there exist at least two iScience Article mechanisms assuring centrosome cohesion, which may explain why enhancing cohesion by overexpressing components related to the proteinaceous linker pathway can overcome deficits observed because of the centrosomal displacement of CDK5RAP2.
We have previously shown that LRRK2-phosphorylated Rab8 and Rab10 and its centrosomal binding partner RILPL1 are required for the process by which LRRK2 causes deficits in centrosomal cohesion (Lara Ordonez et al., 2019;Madero-Pé rez et al., 2018). We show here that the LRRK2-phosphorylated Rab proteins associate with centrosomes, which correlates with the centrosomal displacement of CDK5RAP2 in a manner dependent on the presence of RILPL1. These data support a model in which the phospho-Rab/RILPL1 complex inhibits the proper centrosomal localization of CDK5RAP2, possibly by directly occluding the interaction of CDK5RAP2 with one of its reported centrosomal binding partners such as pericentrin (Pagan et al., 2015). In support of this notion, proximity ligation assays show a pronounced decrease of the CDK5RAP2-pericentrin signal which is reverted by LRRK2 kinase inhibitor.
The precise mechanism by which CDK5RAP2 regulates centrosomal cohesion is still unclear. Its persistence at the centrosome throughout mitosis makes it unlikely that it is part of a cell cycle-regulated linker structure. Rather, it may act in conjunction with pericentrin to regulate pericentriolar matrix and centrosome maturation (Graser et al., 2007;Kim and Rhee, 2014;Lee and Rhee, 2010), or assure additional control of centrosome cohesion via cytoskeletal components (Panic et al., 2015).
Loss of CDK5RAP2 causes reduced proliferation and premature differentiation of neural progenitor cells in vitro and in the intact mouse brain (Buchman et al., 2010;Kraemer et al., 2015;Lizarraga et al., 2010), and it will be interesting to determine whether the reported decrease in adult neurogenesis in LRRK2 G2019S transgenic mice, and the deficits in clonal expansion and differentiation of LRRK2 G2019S iPSCderived neural progenitor cells are related to altered centrosomal CDK5RAP2 localization and concomitant cohesion deficits (Liu et al., 2012;Winner et al., 2011). Centrosomal cohesion deficits do not lead to cellcycle arrest (Faragher and Fry, 2003;Fry et al., 1998a;Mayor et al., 2000), but can cause pronounced deficits in directional cell migration (Flanagan et al., 2017;Floriot et al., 2015), just as we and others have reported for pathogenic LRRK2-expressing cells (Caesar et al., 2013;Choi et al., 2015;Madero-Pé rez et al., 2018). Importantly, future experiments aimed at determining alterations in the localization of CDK5RAP2 as an easy readout for enhanced LRRK2 kinase activity in patient-derived peripheral cells are warranted, as this may allow for the design of high-throughput patient stratification assays in clinical settings when employing LRRK2-related therapeutics.

Limitations of the study
In this study, we have demonstrated the mechanism underlying the pathogenic LRRK2-mediated centrosomal cohesion deficits in various cell types. A limitation of this study is the absence of in vivo data to address the potential pathophysiological relevance of these alterations. However, cohesion deficits are observed in peripheral cells from LRRK2-PD patients, and centrosomal CDK5RAP2 displacement may serve as a proxy for increased LRRK2 kinase activity and amenable to high-throughput patient stratification purposes in clinical settings.

Lead contact
Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Sabine Hilfiker (sabine.hilfiker@rutgers.edu).

Materials availability
Plasmids and cell lines generated in this study are available upon request.

Data and code availability
All immunoblotting and microscopy data reported in this paper will be shared by the lead contact upon request.
This paper does not report original code.
Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

Human iPSC lines and gene editing
Full information about the iPSC lines used in this study can be found in Table S1. The SFC840-03-03 LRKK2wt/R1441C-H3 line was generated by introducing the R1441C mutation on the SFC840-03-03 control line using CRISPR/Cas9 double nickase strategy followed by homology-directed repair with donor sequence. Two gRNAs targeting the desired sequences ( Figure S1A) were cloned into a Cas9 nickase (Cas9-D10A nickase mutant) plasmid with puromycin selection (PX462; Addgene, #62987). Donor DNA template from the SFC840-03-03 control line was cloned into the pGEMâ-T Easy vector (Promega) followed by the introduction of the R1441C mutation, a silent mutation in the PAM region to maximize the efficiency of gene-editing, and two silent mutations to create a unique KpnI restriction site for screening clones ( Figure S1A). SFC840-03-03 iPS cells were nucleofected with the gRNA and donor DNA plasmids and selected with 1 mg/mL puromycin for 4 days. Surviving iPSCs were re-plated as single cells (15'000 cells in a 10 cm dish) and clones were screened by DNA sequencing and KpnI digestion. Positive clones were expanded, re-sequenced ( Figure S1B), karyotyped and screened for a panel of common SNPs ( Figure S1C), tested for pluripotency markers ( Figure S1D) and common iPSC colony morphology ( Figure S1E).

iPSC culture and NPC differentiation
Control and LRRK2 hiPSC lines were cultured in plates previously pre-coated with 1% geltrex (Thermofisher, A1413302) in KnockOut DMEM (Thermofisher, 10829-018) in cell culture media consisting of Essential 8 TM Medium (E8) (Thermofisher, A1517001), and were passaged as colonies with 0.5 mM EDTA (Thermofisher, 15575020) in PBS (Lonza, BE17-516F). For immunocytochemistry, cells were washed with PBS and treated with accutase (Thermofisher, A11105-01) for 5-10 min, and were collected with PBS and 10 mM Y-27632 ROCK inhibitor (Abcam, ab120129). After centrifugation for 5 min at 200 x g, cells were resuspended in E8 in the presence of 10 mM ROCK inhibitor, and were plated at a confluence of 50 0 000 cells/ml onto 1% geltrex pre-coated coverslips. Ciliogenesis was determined from cells grown in serum-free defined E8 iScience Article settings and exposure times were used for image acquisition of individual experiments to be quantified. The average distance between duplicated centrosomes is cell type-dependent. Original studies in U2OS cells determined the distance between duplicated centrosomes to be < 2 mm in 90-95% of cells, and centrosome splitting was defined as cells where the duplicated centrosomes were >2 mm apart (Meraldi and Nigg, 2001). Performing identical analyses, centrosomes were scored as separated when the distance between their centers was >1.5 mm (HEK293T, iPS and NPC), >2.5 mm (A549, primary human dermal fibroblasts), or >5 mm (MEF cells), respectively. In all cases, mitotic cells were excluded from the analysis.
For most experiments, images were analyzed by one or two additional observers blind to condition, with identical results obtained in all cases.
Integrated density determinations were performed over non-saturated images acquired with the same acquisition settings. A circle with diameter of 1.6 mm was placed over each centrosome as identified by pericentrin staining, and integrated density of centrosomal staining determined for each cell, with background intensity adjacent to the centrosome subtracted in all cases. An average of 50-70 individual cells per condition per experiment were quantified in this manner.
For determination of the percentage of ciliated cells, around 200 random cells per experiment were scored for either the absence or presence of primary cilia as assessed using Arl13b staining. Quantification was performed by two observers blind to condition, with identical results obtained in both cases. For determination of proximity ligation assays (PLA), 100-120 random non-transfected or transfected cells were scored for the presence or absence of a perinuclear dot-like PLA signal per condition and experiment by an observer blind to condition.

Centrosome isolation
Centrosomes were isolated from adherent cells using hypotonic cell lysis and purification on sucrose gradients according to the Gogendeau protocol (Gogendeau et al., 2015). Briefly, exponentially growing non-transfected or transfected HEK293T cells (4 3 10 7 cells in two T175 cell culture flasks) were incubated with culture medium containing 1 mg/mL nocodazole and 1 mg/mL cytochalasin D (Sigma, C2618) for 1 h at 37 C to depolymerize microtubule and actin filaments. Cells attached to flasks were gently washed with 10 mL ice-cold PBS, followed by a wash with 10 mL ice-cold 0.1 X PBS containing 8% sucrose, and a wash with 10 mL ice-cold hypotonic lysis buffer (1 mM  iScience Article b-mercaptoethanol, 1 mM Na 3 VO 4 , 5 mM NaF, 1 mM PMSF and protease inhibitor cocktail (Roche, 4693124001). Cell lysis was performed with a total of 5 mL lysis buffer containing 0.5% NP-40 for 10 min at 4 C on a shaker. Lysates were cleared by centrifugation at 2 0 500 x g for 10 min at 4 C, and the supernatant was adjusted to a final 10 mM PIPES, pH 7.2 concentration, layered on top of 1 mL of a 60% sucrose cushion in gradient buffer (10 mM PIPES pH 7.2, 0.1% Triton-X100, 0.1% b-mercaptoethanol), and centrifuged at 10 0 000 x g for 40 min at 4 C. The supernatant was carefully removed, and around 30% of the initial volume collected from the bottom of the tube. This fraction, enriched for centrosomes, was diluted to around 8% sucrose in lysis buffer containing 10 mM PIPES, and loaded on top of a discontinuous sucrose gradient (2 mL 70% sucrose, 1 mL 50% sucrose, 1 mL 40% sucrose in gradient buffer) in ultracentrifuge tubes (Beckman Ultra-Clear, 14 3 95 mm, cat. no. 344060). Centrosomes were isolated by ultracentrifugation at 120 0 000 x g for 2 h at 4 C using an SW40Ti rotor (Beckman), and fractions collected from the bottom using a Beckman recovery system (1 mL for first fraction, and 0.3 mL for all subsequent fractions). Samples were resuspended in 5 x SDS sample buffer, and 20 mL of each fraction resolved by SDS-PAGE and Western blotting with the indicated antibodies.

QUANTIFICATION AND STATISTICAL ANALYSIS
All the statistical details of the experiment can be found in the legend of each figure. For each statistical analysis, at least three independent experiments were carried out. Data were analyzed with a one-way ANOVA and Tukey's post-hoc test, and p < 0.05 was considered significant. The following significance thresholds were used throughout the study: ****p < 0.001; ***p < 0.005; **p < 0.01; *p < 0.05. The values in the bar and line graphs represent the means +/À s.e.m. in all figures.